Bca Compatible Filer

G r a s p t h e P r o t e o m e® Protein Assay ™

The BCA Protein Assay – Reducing Agent Compatible BCA is always compatible with more detergents, buffers/salts and solvents than any other colorimetric protein assay. Now it’s compatible with reducing agents at concentrations routinely used in protein sample buffers! The BCA™ Assay† provides one of the most accurate measurements of protein concentration in biological samples available. Although the BCA™ Assay is compatible with more detergents, buffers/salts and solvents than any colorimetric protein assay, the presence of disulfide reducing agents, including dithiothreitol (DTT) and 2-mercaptoethanol interferes with the assay (Figure 1). The BCA™ Protein Assay Kit – Reducing Agent Compatible† (Product # 23250) from Pierce provides all the advantages of the original BCA™ Assay as well as compatibility with reducing agents at concentrations routinely used in protein sample buffers (Figures 1 and 3). 1

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Figure 1A Standard BCA™ Protein Assay

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Figure 1B BCA™ Protein Assay – Reducing Agent Compatible

Figure 1. Stylized comparison of reducing agent compatibility of standard BCA™ Protein Assay with the BCA™ Protein Assay – Reducing Agent Compatible. Reducing agents (> 1 mM) interfere with the standard BCA™ Protein Assay by artificially increasing the color intensity (Figure 1A). The color intensity produced by the BCA™ Protein Assay – Reducing Agent Compatible is unaffected by the presence of reducing agents (Figure 1B). All samples contained 5 mM DTT and BSA standards at the following concentrations: Tube 1: 0 µg/ml, Tube 2: 125 µg/ml, Tube 3: 250 µg/ml, Tube 4: 500 µg/ml, Tube 5: 750 µg/ml, Tube 6: 1,000 µg/ml, Tube 7: 1,500 µg/ml and Tube 8: 2,000 µg/ml. Step 1. Eliminate reducing agent interference.

Step 2. Add 1 ml BCA™ Working Reagent.

Incubate 15 minutes at 37°C

Step 3. Cool sample 5-10 minutes at room temperature.

Step 4. Read at 562 nm.

Incubate 30 minutes at 37°C

25 µl sample +25 µl Compatibility Reagent in Reconstitution Buffer

Spectrophotometer

Figure 2. BCA™ Protein Assay – Reducing Agent Compatible protocol.

Customer Assistance E-mail: [email protected] • Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316 Outside the United States, visit our web site or call 815-968-0747 to locate your local Perbio Science branch office (below) or distributor Belgium & Dist.: Tel +32 53 85 7184 [email protected]

China: Tel +86 10 8049 9033 [email protected]

France: Tel 0800 50 82 15 [email protected]

Germany: Tel 0228 9125650 [email protected]

Hong Kong: Tel 852 2753 0686 [email protected]

The Netherlands: Tel 076 50 31 880 [email protected]

United Kingdom: Tel 0800 252185 [email protected]

Switzerland: Tel 0800 56 31 40 [email protected]

© Pierce Biotechnology, Inc., 2005. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only. Prices listed herein are accurate at time of printing. For up-to-date prices, visit our web site. BCA™, Coomassie Plus – the Better Bradford™ Assay and Micro BCA™ are trademarks of Pierce Biotechnology, Inc. Triton® is a trademark of Rohm & Haas. Tween® is a trademark of ICI Americas.

Protein Assay Compatibility Table Overview of substances compatible with Pierce Protein Assays* Substance

BCA – Reducing Agent Compatible Detergents (<) Tween®-20 10% Triton® X-114 2% Triton® X-100 10% CHAPS 10% SDS 10% Octyl β-thioglucopyranoside 10% Zwittergent 3-14 2% Reducing Agents Dithiothreitol (DTT) 5 mM 2-Mercaptoethanol 35 mM TCEP 10 mM Chelating Agents EDTA 20 mM Sodium Citrate 100 mM Buffers/Salts Tris 50 mM HEPES, pH 7.5 200 mM MES, pH 6.1 100 mM Imidazole, pH 7.0 50 mM Guanidine•HCl 2M Urea 4M Sucrose 40%

BCA™ Assay (<) 5% 1% 5% 5% 5% 5% 1%

Micro BCA™ Assay (<) 5% 0.05% 5% 1% 5% 5% n/c

Modified Lowry (<) 0.062% 0.031% 0.031% 0.062% 1% n/a n/a

Coomassie (Bradford) (<) 0.062% 0.125% 0.125% 5% 0.125% 3% 0.025%

Coomassie Plus – The Better Bradford™ Assay (<) 0.031% 0.062% 0.062% 5% 0.016% 3% 0.025%

1 mM 1 mM n/c

n/c 1 mM n/c

n/c 1 mM n/a

5 mM 1M n/a

5 mM 1M n/a

10 mM 200 mM

0.5 mM 5 mM

1 mM 0.1 mM

100 mM 200 mM

100 mM 200 mM

100 mM 100 mM 100 mM 50 mM 4M 3M 40%

50 mM 100 mM 100 mM 12.5 mM 4M 3M 4%

10 mM 1 mM 125 mM 25 mM n/a 3M 7.5%

2M 100 mM 100 mM 200 mM 3.5 M 3M 10%

2M 100 mM 100 mM 200 mM 3.5 M 3M 10%

n/a: not assayed n/c: not compatible *Refer to product instruction books for more comprehensive compatibility information 1.2

1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

1.0 0.8 0.6 0.4 – DDT + DDT

0.2 0 0

500

1,000 BSA (µg/well)

1,500

2,000

1.2 10 mM TCEP Net Absorbance (562 nm)

Reference

Net Absorbance (562 nm)

5 mM DDT

Highlights: • Compatible with up to 5 mM DTT, 35 mM 2-mercaptoethanol or 10 mM TCEP • Linear working range: 125-2,000 µg/ml • Sample volume: 25 µl • Compatible with most ionic and nonionic detergents • Significantly less (14-23%) protein:protein variation than coomassie (Bradford)-based methods • Colorimetric method; measure at 562 nm • Easy-to-use protocol (Figure 2)

1.0 0.8 0.6 0.4 – TCEP + TCEP

0.2 0 0

500

Ordering Information 23250

BCA™ Protein Assay Kit – Reducing Agent Compatible Sufficient reagents to perform 250 standard tube assays. Includes: BCA™ Reagent A, 250 ml BCA™ Reagent B, 25 ml Compatibility Reagent, 10 x 20 mg Reconstitution Buffer, 15 ml Albumin Standard (2 mg/ml), 10 x 1 ml ampules

BCA™ Technology is protected by U.S. patent # 4,839,295. Patent pending on Reducing Agent-compatible BCA™ Technology. †

PKG. SIZE

Kit

1,500

2,000

1.2 35 mM 2-Mercaptoethanol 1.0 0.8 0.6 0.4 – 2ME + 2ME

0.2 0 0

500

1,000 BSA (µg/well)

1,500

2,000

Figure 3. BCA™ Protein Assay – Reducing Agent Compatible produces a linear standard curve in the presence of reducing agents. Color response curves for BSA after treatment with Reducing Agent Compatible Reagent in the presence and absence of 5 mM DTT, 35 mM 2-mercaptoethanol and 10 mM TCEP.

www.piercenet.com/brac92h

# 1601288 08/05

DESCRIPTION

Net Absorbance (562 nm)

PRODUCT #

1,000 BSA (µg/well)